Very long chain polyunsaturated fatty acids (VLC-PUFAs), with their structurally unusual long hydrocarbon chains, are essential lipids that play important roles in certain biological systems that cannot be fulfilled by the more common shorter chain C16-C18 fatty acids25. Because of their very long chain structure, some VLC-PUFAs are able to span and reside within both leaflets of the lipid bilayer, thereby giving stability to highly curved cellular membranes such as those which surround nuclear pore complexes25. In photoreceptors, for example, the VLC-PUFAs are known to be associated with rhodopsin and play a role in regulation of phototransduction cascades18,21. Absence of these VLC-PUFAs appears to contribute to macular degeneration in autosomal dominant Stargardt macular dystrophy (STGD3, MIM600110)18, for example.
Three independent mutations in exon six of the Elongation of Very Long chain fatty acids-4 (ELOVL4) gene are associated with STGD31,2. These mutations have been shown to cause truncation and subsequent mislocalization and aggregation of the normal ELOVL4 protein product3-5. Based on sequence homology with a group of functional yeast genes and other mammalian ELOVLs, the ELOVL4 protein was predicted to be involved in elongation of very long chain fatty acids (VLCFAs)1,4,6. For example, the microsomal ELO1 (Ssc1) is responsible for elongation of 14:0 to 16:07,8. ELO2 (Ssc2), ELO3 (Cig30), and ELOVL5 have been shown to be involved in elongation of saturated, monounsaturated, or polyunsaturated fatty acids from 18 to 26 carbons8-10. However, whether the ELOVL4 protein is involved in fatty acid elongation, and the specific step it may catalyze, has remained unknown9,11. Based on its high level of expression in retinal photoreceptor cells, and to lesser extents in brain, testis and skin, it was first hypothesized that ELOVL4 may be involved in the biosynthetic pathway of docosahexaenoic acid (22:6n3, DHA), the most abundant polyunsaturated fatty acid in the retina and the brain1. However, recent experiments carried out in our laboratory (unpublished data) and results obtained from ELOVL4 mutant mice12-14 do not support this hypothesis.
Current reports establish ELOVL4 as an essential protein for growth and development as homozygous ELOVL4 knock-out or knock-in of ELOVL4 mutant genes results in neonatal lethality in mice12-15. The heterozygote knock-in mouse, in which the mutant human ELOVL4 gene has replaced one wild type copy, develops progressive photoreceptor degeneration similar to human STGD3, demonstrating its association with the function of photoreceptors16. Homozygous neonates exhibit scaly wrinkled skin due to severely compromised epidermal permeability barrier and die within hours of birth12-15,17. Lipid analysis of the epidermis from the homozygote ELOVL4 knock-out mice indicated a global reduction in very long chain saturated and hydroxy fatty acids longer than 26:0 in omega hydroxyl ceramides/glucosylceramides and free fatty acid fractions12-15. Also, mice with one normal gene replaced with a gene containing the STGD3 disease mutation had lower amounts of C32-C36 acyl phosphatidylcholines in their retinas18. Based on these findings it was proposed that the ELOVL4 protein functions in VLC-FA and/or VLC-PUFA biosynthesis14,15,18. However, none of the studies published prior hereto have provided direct evidence that ELOVL4 is an elongase. Rather, previous work has only shown that absence or reduced expression of ELOVL4 leads to a reduction in the levels of certain fatty acids. This result could have several explanations not related to elongase activity. For example, ELOVL4 protein could provide some co-factor necessary for VLC-FA/PUFA synthesis or ELOVL4 protein could support activity of a cellular organelle necessary for VLC-FA/PUFA synthesis.
Moreover, research into the VLC-PUFA class of fatty acids has been restricted due to the unavailability of these fatty acids, and their use in commercial formulations and compositions has not been feasible due to the lack of known processes for synthesizing them. It is to the development of such synthetic methods and the compositions and uses of VLC-PUFAs produced therefrom, that the present invention is directed.